Technical Abstract:
: Dimethyladenosine transferase (KsgA) performs diverse roles in bacteria including ribosomal maturation, DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that ksgA mutation in Salmonella Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of ksgA mutant (ksgA::Tn5) of S. Enteritidis in orally challenged one-day-old chickens. The ksgA::Tn5 showed significantly reduced intestinal colonization and organ invasiveness in chickens when compared to the wild-type parent (WT). Phenotype Microarray (PM) was employed to compare ksgA::Tn5 and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C and 42°C. At chicken body temperature (42°C), ksgA::Tn5 showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations =6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, ksgA::Tn5 displayed a severe growth defect in high osmolarity (6.5% NaCl) conditions in nutrient rich (LB) and nutrient limiting (M9 minimum salts) media at 42°C. Moreover, ksgA::Tn5 showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike E. coli, ksgA::Tn5 did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity and altered susceptibility to kasugamycin and chloramphenicol.